Biofilm-producing and specific antibiotic resistance genes in Pseudomonas aeruginosa isolated from patients admitted to a tertiary care hospital, Bangladesh

Highlights • Biofilm-forming genes are useful to identify biofilm in Pseudomonas aeruginosa.• Biofilm is not necessary for antibiotic resistance in P. aeruginosa.• The pqsA gene is the most prevalent biofilm-forming gene in P. aeruginosa.


Introduction
Pseudomonas aeruginosa ( P. aeruginosa ) has various factors that help to adhere and damage cells, and mucosal tissues of the host, elicit inflammation, and impair defense mechanisms [ 1 ].Biofilm is one of the factors that help in the establishment of the organism on different host tissues, especially in immunocompromised patients, patients with implanted devices, and burn wounds [ 2 , 3 ].
Biofilm is a network among multilayered cell clusters that acts as a protective barrier against the host immune system and antibiotic therapy [ 4 ].P. aeruginosa produces three extracellular polysaccharides (EPS): alginate, polysaccharide (Psl), and pellicle), that help in biofilm formation [ 5 ].Quorum sensing (QS), a cell-to-cell signaling system, aids the production of different virulent factors causing chronic infection [ 6 ].The pqs QS system uses Pseudomonas quinolone signal (PQS) as the signal molecule acting as an ideal anti-virulence drug target [ 7 ].In the Las QS system, LasR (elastase) gene acts as a transcriptional activator that encodes virulence factors like protease, elastase, hydrogen cyanide, and phenazines [ 8 ].
Tolerance to killing by antimicrobials is the hallmark of biofilm [ 9 ].P. aeruginosa uses a dual resistance mechanism: reduced penetration and active drug efflux [ 10 , 11 ].Moreover, bacteria embedded in the biofilm develop tolerance against high antibiotic concentrations [ 12 ].The ndvB gene encodes glucosyltransferase that is involved in the synthesis of cyclic glucagon situated at the periplasm.P. aeruginosa cyclic glucans interact with antibiotics and sequester them in the periplasm [ 13 ].Moreover, P. aeruginosa also contains adenosine triphosphate-binding cassette (ABC) transport system [ 14 ].ABC transport system includes PA1874, PA1875, PA1876, and PA1877 (PA1874-77) that remove antibiotics from the cells within the biofilm [ 9 ].
Young biofilms are more susceptible to antibiotics compared to more developed biofilms [ 15 ].Thus, early intervention could be useful rehttps://doi.org/10.1016/j.ijregi.2024.100369garding the management of intractable biofilm-associated persistent infections.Very few studies evaluated the accuracy of different methods for the detection of biofilm.Molecular methods are more sensitive and specific.However, no study on the genes responsible for biofilm formation and biofilm-associated antimicrobial resistance among clinical isolates of P. aeruginosa in Bangladesh.Therefore, this study was conducted to detect biofilm, and genes responsible for biofilm and antibiotic resistance among P. aeruginosa.

Sample collection
This cross-sectional study was conducted in the Department of Microbiology of Dhaka Medical College (DMC), Dhaka, Bangladesh, from July 2017 to December 2018.Among 446 samples (infected burn, surgical wounds, and endotracheal aspirate [ETA]), a total of 232 clinical isolates of Pseudomonas aeruginosa from the infected burn, surgical wounds, and ETA were included.

Bacterial isolation
After collecting aseptically, samples were inoculated in blood agar media and MacConkey agar media followed by incubation aerobically at 37°C and 42°C for 48 hours.P. aeruginosa was isolated and identified by colony morphology, Gram staining, and biochemical tests following standard procedures [ 16 ].Identification of P. aeruginosa was confirmed by polymerase chain reaction (PCR) from the culture with specific primers [ 17 ].

Antimicrobial susceptibility testing
Isolates were tested for antimicrobial susceptibility using Kirby-Bauer modified disk diffusion method and agar dilution method of minimal inhibitory concentration [ 18 ].Antibiotic susceptibility was interpreted following Clinical and Laboratory Standards Institute guideline, 2017 [ 19 ].

Method of detection of biofilm
Congo red agar (CRA) method: CRA medium was prepared with brain heart infusion broth 37 g/L, sucrose 50 g/L, agar No. 1 10 g/L, and Congo red indicator eight g/L.Congo red stain and brain heart infusion agar with sucrose were autoclaved separately followed by mixing at 55°C.CRA plates were inoculated with test organisms and incubated at 37°C for 24 hours aerobically.Black colonies with a dry crystalline consistency indicated biofilm production [ 20 ].The experiment was performed in triplicate and repeated three times.

Tube method
A loopful of test organisms was inoculated in 10 mL of Trypticase Soy Broth with 1% glucose in test tubes.The tubes were incubated at 37°C for 24 h.After incubation, tubes were decanted and washed with phosphate-buffered saline (PBS) (pH 7.3) and dried.Tubes were then stained with crystal violet (0.1%).The excess stain was washed with deionized water and dried in the inverted position.The scoring for the tube method was done according to the results of the control strains.Biofilm formation was considered positive when a visible film lined the wall and the bottom of the tube.The amount of biofilm formed was scored as 1-weak/none, 2-moderate, and 3-high/strong.The experiment was performed in triplicate and repeated three times [ 21 ].

Tissue culture plate method
Cultures were transferred into a fresh medium in a dilution of 1:100.Ninety-six well flat bottom polystyrene treated tissue culture plates (TCP) (SigmaAldrich, Costar, USA) were filled with 200 μL of diluted cultures.Control organisms were also incubated, diluted, and added to the TCP.The sterile broth was taken as a negative control.After incubation at 37°C for 24 hours, the contents of each well were removed by gentle tapping and washed with 0.2 mL PBS four times to remove free-floating bacteria.Biofilm formed by bacteria adherent to the wells was fixed by 2% sodium acetate and stained by crystal violet (0.1%).The excess stain was removed with deionized water and dried.Optical density (OD) of stained adherent biofilm was obtained using a micro-ELISA auto reader (model 680, Biorad, UK) at wavelength 570 nm.The experiment was performed in triplicate and repeated three times [ 22 ].

Calculation of OD values
The average OD values were calculated for all tested strains and negative controls since all tests were performed in triplicate and repeated three times.Second, the cut-off value (ODc) was established.It was defined as three standard deviations (SD) above the mean OD of the negative control: ODc = average OD of negative controls + (3 × SD of negative control) [ 23 ].It was interpreted as, if average OD ≤ ODc, no biofilm was produced, if ODc < OD ≤ 2ODc, weak biofilm was produced, 2ODc < OD ≤ 4ODc, moderate biofilm was produced, if 4ODc < OD, then high biofilm was produced [ 23 ].

Polymerase chain reaction
Deoxyribonucleic acid (DNA) extraction: Bacterial pellets were mixed with 300 l distilled water followed by boiling at 100°C for 10 minutes in a block heater (DAIHA Scientific, Seoul, Korea).After cooling on the ice pack, the mixture was centrifuged at 4°C at 13,500g for 10 minutes.The extracted DNA was then kept at -20°C [ 24 ].
Amplification: After making a 25 l mixture of mastermix, primer, and DNA template, PCR was performed in DNA thermal cycler (Eppendorf AG, Mastercycler gradient, Hamburg, Germany).The amplified DNA was analyzed by 1.5% agarose gel-electrophoresis at 100 volts for 35 minutes, stained with 1% ethidium bromide, and visualized under an ultraviolet transilluminator (Gel Doc, Major science, Taiwan).

Data analysis
Data analysis was done by using 'Microsoft Office Excel 2010' program.The test of significance was calculated using chi-square test, and P value < 0.05 was taken as minimal level of significance.Source: Original.ETA, endotracheal aspirate; N, total number of samples; n, number of P. aeruginosa having respective biofilm-producing genes.

Results
A total of 446 samples were collected, among which 232 (52.02%) yielded growth of P. aeruginosa .Detection of biofilm production in different methods by these P. aeruginosa from different samples is shown in Table 1 .
Considering TCP as the gold standard method, comparisons of diagnostic parameters (sensitivity, specificity, positive and negative predictive value, and accuracy) between TM and CRA methods are demonstrated in Table 2 .
In Table 3 , the distribution of biofilm-forming genes among 24 biofilm-producing isolates in the TCP method is shown.pqsA was the most common gene isolated from biofilm-forming P. aeruginosa isolated from both burn and ETA samples, followed by pslA and pelA .
Antibiotic resistance pattern of all the isolated P. aeruginosa (N = 232) , biofilm-forming (N = 24), and non-biofilm-forming (N = 208) is demonstrated in Table 4 .Though a higher percentage of resistance was observed to most antibiotics among biofilm-forming stains, resistance to cefotaxime was statistically significant in non-biofilm-forming strains, whereas resistance to colistin was statistically significant among biofilm-forming P. aeruginosa ( Table 4 ).

Discussion
Biofilm-related infections are subject to develop recurrent and chronic wound infections, and device-related infections.Besides, b Resistance to Cefotaxime is statistically significant in biofilm negative P. aeruginosa .biofilm-related bacterial infection increases mortality rate in burn patients [ 30 ].
Detection of biofilm is crucial for the prevention of treatment failure, as it is a threat to hospital-acquired infections [ 31 ].Although the molecular method is more sensitive and specific, phenotypic methods are comparatively less expensive and easier to perform.Sultan and Nabiel reported higher sensitivity and specificity in the tube method, which contradicted our study [ 32 ].In this study, considering TCP method as the gold standard, the tube method showed cent per cent negative pre-dictive value, and the CRA method displayed 100% positive predictive value.Sultana found a higher percentage of biofilm production in Dhaka Medical College and Hospital (61.54% in TCP, 50% in TM, 11.84% in CRA method) [ 33 ].There is no clear explanation for such variations in these studies, but it can be due to batch-to-batch variation of media and subjective judgment of interpretation as TM and CRA are qualitative tests [ 32 ].
Biofilms adhere to the human cell surface as a community of microorganisms.These organisms are embedded in the matrix of EPS, selfproduced by these adherent cells.pel and psl operons biosynthesize the EPS and help in the interactions between cells during biofilm formation [ 34 ].In current study, biofilm-forming gene pqsA (79.17%) was the most prevalent in P. aeruginosa isolated from both burn wound and ETA samples.The presence of pslA (70.83%), pslD (45.83%), pslH (37.5%) in biofilm-forming P. aeruginosa isolated from burn wound samples was statistically significant.Rajabi et al. reported significant association of psl and pel genes with biofilm formation [ 35 ].Furthermore, LasR is a major regulator of QS system in P. aeruginosa.However, LasR gene and pqsA gene do not necessarily co-exists [ 36 ].In this study, LasR gene was less prevalent among biofilm-forming strains.Though biofilm formation is a multistage process that is attributed to many factors, these genes may act as markers for screening biofilm-forming bacteria.
The resistance in biofilms is multifactorial, including the diffusion barrier and efflux transporter in P. aeruginosa [ 37 ].Zhang et al. described mutations within the ndvB gene of P. aeruginosa , which encodes a glucosyltransferase, resulting in increased sensitivity of P. aeruginosa biofilms to several antibiotics [ 38 ].In this study, 16.67% of biofilmforming P. aeruginosa were positive for biofilm-specific antimicrobial resistance gene ndvB , followed by 8.33% for PA1874 and PA1877, which are not substantial.Besides, resistance to cefotaxime was statistically significant in non-biofilm-forming P. aeruginosa compared to biofilmforming strains.In contrast, colistin resistance among biofilm-forming P. aeruginosa was statistically significant (p < 0.05) compared to nonbiofilm-forming isolates.So, it was inconclusive whether there was association between biofilm formation and antibiotic resistance in P. aeruginosa in this study.Because resistance patterns of other antibiotics among biofilm-forming and non-biofilm-forming P. aeruginosa isolated in this study were almost similar.A lower number of biofilm-forming isolates could be responsible for this matter.

Conclusions
Biofilm-forming Pseudomonas aeruginosa was detected from infected burn wounds and ETA samples by TCP method.Detection of biofilmforming genes can assist in screening biofilm-producing bacteria.Both biofilm-forming and non-biofilm-forming P. aeruginosa were resistant to common antibiotics.ndvB, PA1874, PA1876, and PA1877 may have a role in antibiotic resistance.However, biofilm-specific antibiotic resistance encompasses multiple mechanisms.Due to time and resource constraints, other mechanisms of antibiotic resistance related to biofilm could not be evaluated.Furthermore, the biofilm detection methods of bacteria were conducted in vitro , and it is still unknown what will occur if biofilm is present in vivo .Nevertheless, biofilm detection in routine laboratory tests could minimize nosocomial infections.It will help the clinicians to select the proper antibiotics for appropriate patients.

Table 1
Detection of biofilm production in different methods by P. aeruginosa from different samples (N = 232).

Table 2
Diagnostic parameters of tube method and Congo red agar method for biofilm detection.

Table 3
Proportion of biofilm-forming genes among biofilm-forming P. aeruginosa detected by tissue culture plate method.
a Resistance to Colistin is statistically significant in biofilm positive P. aeruginosa .